The early diagnosis of cystinosis and Wilson disease (WD) is critical, because effective treatments exist. Unfortunately, most patients with cystinosis or WD are diagnosed after developing significant complications; in particular, kidney, liver and brain damage. There are no cost-effective screening methods available for either disorder. Many congenital disorders such as Primary Immunodeficiency diseases (PIDDs), cystinosis, and WD are caused by mutations that result in absent or significantly diminished levels of proteins; thus, protein biomarkers localized within the cells (either cytoplasm or transmembrane) have enormous potential in the diagnosis/screening of congenital disorders. We believe that LC-MRM-MS based approach makes sense as a rapid, inexpensive approach to simultaneously screen for a variety of congenital disorders. As proof-of- concept, we have demonstrated that LC-MRM-MS analysis of signature peptides can identify patients lacking specific protein markers of three life-threatening PIDDs: SCID, WAS, and XLA using White Blood Cells (WBC). However, sensitivity was constrained by sample complexity, especially for these low abundance proteins to be detected/quantified directly from DBS without enrichment. Extensive studies have showed that the limit of sensitivity can be improved by peptide immunoaffinity enrichment coupled to MRM (immuno-MRM), which achieves more than a 1000-fold enrichment for target peptides. Our goal is to develop and validate a specific and quantitative assay that will simultaneously identify multiple genetic conditions using a small volume of blood including DBS. The objective of this application is to develop a LC-MRM-MS based approach to quantify a panel of biomarkers in infant blood to facilitate the early detection and diagnosis of the two congenital disorders, cystinosis and WD. Our specific aims are to: 1: Identify proteotypic peptides for Cystinosin (CTNS), Sedoheptulokinase (SHPK) and ATP7B; marker proteins for Cystinosis and Wilson diseases. We hypothesize that we can quantify low abundance proteins by LC-MRM-MS method based on our preliminary data. We will test our hypothesis first by examining human cell lines to select proteotypic signature peptides for these two diseases and fully optimizing LC-MRM-MS conditions. 2. Increase sensitivity of the MRM assay for the two congenital disorders by coupling it with peptide immunoaffinity enrichment. With peptide immunoaffinity enrichment, we hypothesize that signature peptides can be quantified simultaneously from a small volume of blood and ultimately blood spots, eliminating the need for isolation of white blood cells. We will test our hypothesis by examining the performance metrics for each assay by generating response curves. 3. Evaluate the ability of multiplex immuno-MRM approach to correctly identify patients with cystinosis or Wilson disease. We hypothesize that the immuno-MRM method will be able to correctly identify patients with cystinosis or Wilson disease in which the target proteins are absent or significantly reduced. We will test our hypothesis by analyzing clinical samples retrospectively.